The Preparation And In Vitro Research Of Liver Target Glycosylated Micelles As Multifunctional Theranostic Nanocarriers For Drug Delivery And MR Imaging

Object1.We tried to prepare a novel multi-functional polymer nanocomposite micelles containing lactose or galactose for MR diagnostics and antineoplastic drug intelligent release,liver active targeting,PCL₃₇-SS-PGluLac10@SPIO/Dox and PCL₃₇-SS-PGluGal₁₀@SPIO/Dox,then characterize its physicochemical properties.2.Through the in vitro cell experiment,we studied the mechanism of micelles uptake and the killing of tumor cells,and the targeting of hepatoma cells was examined.The ability of in vitro tumor cell magnetic resonance imaging was confirmed by in vitro MRI imaging.MethodsFirst with dodecanol as initiator,?PCL?was prepared by ring-opening polymerization of caprolactone??-CL?.The hydroxyl groups of PCL were then converted to a disulfide-linked amino group?PCL-ss-NH2?by a two-step chemical reaction,and then PCL-ss-NH2 was used as macromolecule initiator to initiate the polymerization of azide glutamic acid ester cyclic anhydride?AELG-NCA?monomer,and the disulfide-containing block polymer PCL-SS-PAELG was successfully prepared.Finally,the alkynylated lactose or alkynylated galactose was coupled to the synthesized copolymer by the "Click" reaction to form PCL₃₇-SS-PGluLac10 and PCL₃₇-SS-PGluGal₁₀.The resulting glycosylated polymer has amphiphilic behavior and can be nanocomposite in self-assembly behavior in aqueous solution.Based on the amphiphilic behavior of the glycosylated block polymer,hydrophobicsuperparamagnetic iron oxide?SPIO?and antitumor drug doxorubicin?DOX?were encapsulated in the hydrophobic core by dialysis method to prepare the tumor theranotics micelles PCL₃₇-SS-PGluLac10@SPIO/Dox and PCL₃₇-SS-PGluGal₁₀@SPIO/Dox.PCL₃₇-SS-PGluLac10 and PCL₃₇-SS-PGluGal₁₀ were characterized by ¹HNMR FTIR.The surface morphology,particle size and diameter distribution of the micelles were determined by DLS and TEM.The critical micelle concentration was measured by pyrene fluorescence method.The bioactivity of the micelles was examined by the specific identification experiment of the plant agglutinin labeled with the fluorescent dye.And the loading rate of dox and SPIO,in vitro drug reduction response intelligent release behavior were studied.The tumor cell lethality of the carbohydrate and drug micelles PCL₃₇-SS-PGluLac10@SPIO/Dox and PCL₃₇-SS-PGluGal₁₀@SPIO/Dox was evaluated by MTT assay.The selected cells were human hepatocellular carcinoma cells?HepG2?.The cell viability was calculated from the absorbance values?OD values?of the different concentrations of wells.Cellular uptake and distribution of polymeric micelles was analyzed by Dox fluorescence,DAPI nuclear staining and flow cytometry.The hepatic targeting of glucose-treated micelles was detected by galactose inhibition assay.The T2 contrast effect of micelles was analyzed by vitro MRI imaging.Results1.¹HNMR and FTIR confirmed that PCL₃₇-SS-PGluLac10 and PCL₃₇-SS-PGluGal₁₀ were successfully prepared.The average particle size of PCL₃₇-SS-PGluLac10@SPIO/Dox was?120.8±1.7?nm,and the average particle size of PCL₃₇-SS-PGlu Gal₁₀@SPIO /Dox was?150.1±2.6?nm.Under the observation of the resulting micelles were uniform spherical structure.2.The CMC value of PCL₃₇-SS-PGluGal₁₀ was 0.0105 mg / mL and the CMC value of PCL₃₇-SS-PGluLac10 was 0.0166 mg / mL.Plant lectin-specific binding experiments show that sugar peptides can specifically bind lectins.DOX release experiments showed that the two micelles were able to release the drug quickly in the presence of GSH,with 58.6% and 70% release at 12 h respectively;almost completely released at 24 h.3.The cell survival rates of PCL₃₇-SS-PGluLac10 and PCL₃₇-SS-PGluGal₁₀ was were maintained at 80% or more after 48 h incubation with HepG2 cells,which provedthat the biosafety of the material was good.The results of cytotoxicity test showed that the survival rate of cells was inversely proportional to the concentration of drugs.With the increase of Dox concentration,the inhibitory effect of cell growth was significant.4.Fluorescence observation,DAPI staining results show that two kinds of glycosylated nano-medical micelles can be quickly into the cytoplasm,Free Dox mainly into the nucleus.In vitro cell cell experiments further demonstrated the targeting of glucose-containing micelles to hepatocarcinoma cells.5.In vitro MRI imaging results showed that PCL₃₇-SS-PGluLac10@SPIO/Dox and PCL₃₇-SS-PGluGal₁₀@SPIO/Dox micelles MR T2 WI signal were gradually decreased with the increase of Fe ion concentration;Respectively 168.1 Fe mM-1 · s-1,259.4 Fe mM-1 s-1,significantly better than Feridex??111.5 Fe mM-1 s-1?.Proved its excellent T2 contrast effect.Conclusion1.In this study,we successfully prepared a new type of amphiphilic polymer nanocomposite micelles containing lactose or galactose for MR diagnostics and antineoplastic drug release,liver targeting,PCL₃₇-SS-PGluLac10@SPIO/Dox and PCL₃₇-SS-PGluGal₁₀@SPIO/Dox.2.The two kinds of sugar-treated micelles were small and uniform in size,and they had good water solubility,colloidal stability,biocompatibility,anti-tumor effect and MR imaging,and realized the active integration of liver targeted diagnosis and treatment.